The medial prefrontal cortex is a primary brain mediator of stress and mood. In humans and in animal models, medial prefrontal cortical dysfunction is associated with emotional disturbances, impaired fear extinction and inefficient termination of physiological stress responses. Medial prefrontal cortex dysfunction is linked to numerous mental illnesses, the most prominent being depression and post-traumatic stress disorder, diseases that are triggered by life stress and result in long-term inappropriate stress responding. Notably, gene expression in the prefrontal cortex is exquisitely sensitive to stress exposure, with the vast majority of regulated mRNAs showing pronounced down-regulation. Recent studies have convincingly demonstrated that non-coding RNAs, including microRNAs and alternatively expressed 3'-unstranslated (3'-UTR) mRNA sequences, play a major role in mRNA down-regulation in numerous tissues, including brain. This Exploratory Proposal is designed to perform detailed analysis of prefrontal cortical non-coding RNAs (microRNAs) and 3' UTRs (mRNAs) using newly-developed deep sequencing technology, affording a heretofore unprecedented assessment of miRNA and 3-UTR regulation by chronic unpredictable stress in rat. The unpredictable stress regimen reliably models physiological and behavioral symptoms of depression, allowing for extrapolation of preclinical findings to putative mechanisms of functional dysregulation in human cortex. Aim 1 will use deep sequencing methods to provide a comprehensive and quantitative analysis of existing as well as novel chronic stress-regulated miRNAs in the prefrontal cortex of C57BL6 mice. Aim 2 will apply the deep sequencing methods to identification of chronic stress-regulated 3'-UTR sequences. In both Aims, follow-up studies will verify specific regulation of targeted miRNAs and 3'-UTR sequences in the prefrontal cortex, and use anatomical methods to localize expression to distinct cortical subregions and cell types. Identification of novel stress-regulated miRNAs and 3'-UTRs in mouse will inform our understanding of mechanisms underlying human stress-related disease, and provide possible future targets for intervention in disease processes.